1. Field of the Invention
The present invention relates to a method of enzymatically measuring hydrogen peroxide with high sensitivity and a reagent therefor. The method is utilized in the field of clinical examinations to quantitatively measure various biological substances and enzymes in a body fluid as a sample, through the measurement of hydrogen peroxide.
2. Related art
In the field of clinical examinations, at the present time, the enzymatical measurement of hydrogen peroxide has widely been carried out. For quantitatively measuring a biological substance or enzyme in a sample body fluid such as serum and urine, such an enzymatical measuring method has been developed and actually employed, which comprises steps of generating hydrogen peroxide in the sample with use of an oxidase, and leading into a color developing reaction with use of an oxidative color developing reagent and an oxidase of peroxidase, catalase or the like.
The following illustrates relations between the biological substances to be quantitatively measured by utilizing the generation of hydrogen peroxide and exemplar oxidases to be used therefor.
a) Activity of amylase: .alpha.-Glucosidase and glucose oxidase. PA1 b) Activity of guanase: xanthine oxidase and uricase. PA1 c) Glucose: Glucose oxidase. PA1 d) Creatinine: Creatininase and safcosine oxidase. PA1 e) Activity of choline esterase: Choline oxidase. PA1 f) Cholesterol: Cholesterol oxidase. PA1 g) Cholesterol ester: Cholesterol ester hydrogenase and cholesterol oxidase. PA1 h) Neutral fat: Lipase and glycerol oxidase. PA1 i) Activity of transaminase: Pyruvate oxidase. PA1 j) Free (or non-esterified) fatty acids: Acyl-CoA synthetase and Acy-CoA oxidase. PA1 k) Lactic acid: Lactate oxidase. PA1 l) Uric acid: Uricase. PA1 m) Urea: Urease and glutaminate oxidase. PA1 n) Phospholipids: Phospholipase D and choline oxidase. PA1 o) Bilirubin: Billrubin oxidase.
The following illustrates color reaction utilizing the generated hydrogen peroxide.
A) Methods using peroxidase (POD): ##STR1##
B) Methods using catalase: There are methods, for instance, wherein methanol is oxidized with generated hydrogen peroxide to be converted into formaldehyde, and then acetylacetone and an ammonium salt are added to cause a condensation according to the Hantzsch reaction and to develop a color. This method can be shown with reaction formulae, as given below. ##STR2##
The latter methods using catalase not only require a relatively long operation period of time but also are difficult to apply using an automatical measuring apparatus. Thus, the former methods using POD have widely been employed in the clinical examination field.
Among the color reactions using POD, the method most widely been accepted is that wherein a hydrogen donor is subjected to a condesation reaction with a coupler such as 4-aminoantipyrine (hereinafter referred to as "4-AA"), 3-methyl-2-benzothiazolinonehydrazone (hereinafter referred to as "MBTH") or the like, in the presence of POD, and then the reaction mixture is lead into an oxidative color developing step.
In this case, the following can be used as the hydrogen donor: phenol, 4-chlorophenol, 2,4-dichlorophenol and sulfonated derivatives thereof, 2,4-dibromophenol, 2,6-dichlorophenol, 2,4,6-tribromophenol, 3,5-dichloro-2-hydroroxybenzene sulfonic acid, 3-hydroxy-2,4,6-triiodobenzoic acid and the like phenols; N,N-dimethylanillne, N,N-diethylaniline, N-ethyl-N-(2-hydroxyethyl)-m-toluidine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N,N-dimethyl-m-anisidine, N-substituted anilines treated with alkylsulfonic acid or hydroxyalkylsulfonic acid or the like anilines.
Advantages and disadvantages of the conventional methods are as follows.
The methods using 4-AA as the coupler have widely been used, since a detection wave-length for dyes lies at a shorter side, it can be applied in a wide pH range, and reagents show good stability, although a sensitivity thereof is lower than that of the method using MBTH as the coupler. If a reducing substance such as reduced type nicotinamidonucleotide or the like coexists in the reaction system, however, the reducing action is apt to be affected due to such a substance, so that it can happen that color does not appear or tone of the color suppressed.
The methods using MBTH as the coupler are known to be suitable for increased; sensitivity, since a sensitivity is high, detection wave-length lies at higher side, developed color shows good stability, and it is not susceptible to influence of the reducing substance, even if the substance coexists in the reaction system. However, there are disadvantages in that the MBTH reagent per se is not stable, a reagent blank value remarkably changes with the lapse of time, and it can be applied for only in acidic pH range, in practice.